PROJECT SUMMARY Antibodies diversify through two distinct pathways. The first involves combinatorial assembly of Immunoglobulin (Ig) variable region (V) exons during B cell development. The second involves V exon somatic hypermutation (SHM) and affinity-based selection in germinal centers (GCs). There are fundamental gaps in understanding how these systems collaborate to recognize, adapt, and neutralize diverse pathogenic threats. The long-term goal is to shed light onto fundamental GC B cell biology and elucidate underlying mechanisms of protective antibody development. The objective for this proposal is to elucidate mechanisms underlying GC plasticity?in particular, the extent of GC diversification, and the parameters that allow B cell GC entry and continued antibody evolution. The central hypothesis is that the GC system provides dual function with regard to antibody development. On one hand, the GC reaction intensifies affinities readily available from the primary repertoire. On the other hand, GC plasticity is flexible enough to allow for the recruitment of extremely low affinity/non-cognate B cell clones whose BCR is not initially of sufficient affinity to compete well in the GC, but which may have unique potential (given a few needed mutations) to recognize critical epitopes not otherwise targeted well by the primary repertoire. A deeper understanding of how these GC functions are regulated promises to reveal new insights into how to more effectively recruit low frequency/low affinity B cells with potential to become broadly neutralizing, and shepherd them toward protective efficacy. This hypothesis will be explored with two specific aims: 1) Characterize the capacity and limitations of diversity mediated by the GC SHM diversification system; and 2) Define features that regulate B cell participation in germinal centers. Under the first aim, the flexibility of affinity development and specificity potential by the GC diversification system will be examined using an extremely low BCR/antigen affinity (Ka<102 M-1), and BCR-negative model systems in the physiologic context of competitive settings. Under the second aim, modifiable factors that regulate the flexibility of SHM-mediated Ig evolution in physiologic contexts will be defined. The approach is innovative, because the applicant's recent published work and preliminary data indicate that GC-mediated diversification can provide specificities to new epitopes not otherwise present in the primary Ig repertoire within a physiologic, competitive environment of a diverse primary Ig repertoire. Innovative mouse models will be used to probe the parameters and mechanistic aspects of the roles of affinity and Ig frequency on participation in GC maturation and contribution to protective antibody responses in the context of an animal model expressing a diverse human Ig repertoire. Discovering how extremely low affinity B cell clones gain access to the GC and continue to mature in the highly competitive GC environment?often accumulating extremely high levels of SHM not yet replicated with vaccines?would be a significant contribution to fundamental B cell knowledge. Such knowledge also has the potential to inform strategies for vaccination as well as antibody bioengineering.